Multiplexed and microparticle-based analyses: quantitative tools for the large-scale analysis of biological systems.

While the term flow cytometry refers to the measurement of cells, the approach of making sensitive multiparameter optical measurements in a flowing sample stream is a very general analytical approach. The past few years have seen an explosion in the application of flow cytometry technology for molecular analysis and measurements using microparticles as solid supports. While microsphere-based molecular analyses using flow cytometry date back three decades, the need for highly parallel quantitative molecular measurements that has arisen from various genomic and proteomic advances has driven the development in particle encoding technology to enable highly multiplexed assays. Multiplexed particle-based immunoassays are now common place, and new assays to study genes, protein function, and molecular assembly. Numerous efforts are underway to extend the multiplexing capabilities of microparticle-based assays through new approaches to particle encoding and analyte reporting. The impact of these developments will be seen in the basic research and clinical laboratories, as well as in drug development.     

Accessing genomic information: alternatives to PCR

The growing abundance of genomic sequence data invites increasingly large-scale genetic analyses. Studies of genetic variation in large sets of genes can illuminate important disease mechanisms and serve to identify novel drug targets or predict therapeutic responses. At present mostly a concern in extensive research projects, large-scale genetic analyses will gradually also find their way into clinical practice as an aid to the physician. It is timely, therefore, to take stock of methods that are becoming available for analyses of large sets of gene sequences. Clearly PCR remains the workhorse for molecular genetic analysis, and several modifications such as homogenous amplification assays and parallel detection on DNA microarrays further increase throughput. Recent developments, however, also offer hope that other methods will become available for genomic investigations, providing substantially increased analytical capacity.     

Parallel gene analysis with allele-specific padlock probes and tag microarrays

Parallel, highly specific analysis methods are required to take advantage of the extensive information about DNA sequence variation and of expressed sequences. We present a scalable laboratory technique suitable to analyze numerous target sequences in multiplexed assays. Sets of padlock probes were applied to analyze single nucleotide variation directly in total genomic DNA or cDNA for parallel genotyping or gene expression analysis. All reacted probes were then co-amplified and identified by hybridization to a standard tag oligonucleotide array. The technique was illustrated by analyzing normal and pathogenic variation within the Wilson disease-related ATP7B gene, both at the level of DNA and RNA, using allele-specific padlock probes.     

Suspension array technology: new tools for gene and protein analysis.

Flow cytometry has long been a key tool in the analysis of lymphocytes and other cells, owing to its ability to make quantitative, homogeneous, multiparameter measurements of particles. New developments in illumination sources, digital signal processing and microsphere chemistry are driving the development of flow cytometry in new areas of biomedical research. In particular. the maturation of approaches to perform highly parallel analyses using suspension arrays of microspheres with different morphospectral features is making flow cytometry an important tool in protein and genetic analysis. In this paper, we review the development of suspension array technology (SAT), current applications in protein and genomic analysis, and the prospects for this platform in a variety of large scale screening applications.     

Pash 3.0: A versatile software package for read mapping and integrative analysis of genomic and epigenomic variation using massively parallel DNA sequencing

Background  

Massively parallel sequencing readouts of epigenomic assays are enabling integrative genome-wide analyses of genomic and epigenomic variation. Pash 3.0 performs sequence comparison and read mapping and can be employed as a module within diverse configurable analysis pipelines, including ChIP-Seq and methylome mapping by whole-genome bisulfite sequencing.     

The use of capillary electrophoresis for DNA polymorphism analysis

Capillary electrophoresis has advanced enormously over the last 10 yr as a tool for DNA sequencing, driven by the human and other major genome projects and by the need for rapid electrophoresis-based DNA diagnostic tests. The common need of these analyses is a platform providing very high throughput, high-quality data, and low process costs. These demands have led to capillary electrophoresis machines with multiple capillaries providing highly parallel analyses, to new electrophoresis matrices, to highly sensitive spectrofluorometers, and to brighter, spectrally distinct fluorescent dyes with which to label DNA. Capillary devices have also been engineered onto microchip formats, on which both the amount of sample required for analysis and the speed of analysis are increased by an order of magnitude. This review examines the advances made in capillary and chip-based microdevices and in the different DNA-based assays developed for mutation detection and genotype analysis using capillary electrophoresis. The automation of attendant processes such as for DNA sample preparation, PCR, and analyte purification are also reviewed. Together, these technological developments provide the throughput demanded by the large genome-sequencing projects.     

MBD-isolated Genome Sequencing provides a high-throughput and comprehensive survey of DNA methylation in the human genome

DNA methylation is an epigenetic modification involved in both normal developmental processes and disease states through the modulation of gene expression and the maintenance of genomic organization. Conventional methods of DNA methylation analysis, such as bisulfite sequencing, methylation sensitive restriction enzyme digestion and array-based detection techniques, have major limitations that impede high-throughput genome-wide analysis. We describe a novel technique, MBD-isolated Genome Sequencing (MiGS), which combines precipitation of methylated DNA by recombinant methyl-CpG binding domain of MBD2 protein and sequencing of the isolated DNA by a massively parallel sequencer. We utilized MiGS to study three isogenic cancer cell lines with varying degrees of DNA methylation. We successfully detected previously known methylated regions in these cells and identified hundreds of novel methylated regions. This technique is highly specific and sensitive and can be applied to any biological settings to identify differentially methylated regions at the genomic scale.     

Detecting cancer gene networks characterized by recurrent genomic alterations in a population

High resolution, system-wide characterizations have demonstrated the capacity to identify genomic regions that undergo genomic aberrations. Such research efforts often aim at associating these regions with disease etiology and outcome. Identifying the corresponding biologic processes that are responsible for disease and its outcome remains challenging. Using novel analytic methods that utilize the structure of biologic networks, we are able to identify the specific networks that are highly significantly, nonrandomly altered by regions of copy number amplification observed in a systems-wide analysis. We demonstrate this method in breast cancer, where the state of a subset of the pathways identified through these regions is shown to be highly associated with disease survival and recurrence.     

Application of resequencing microarrays in microbial detection and characterization

Microarrays are powerful, highly parallel assays that are transforming microbiological diagnostics and research. The adaptation of microarray-based resequencing technology for microbial detection and characterization resulted in the development of a number assays that have unique advantages over other existing technologies. This technological platform seems to be especially useful for sensitive and high-resolution multiplexed diagnostics for clinical syndromes with similar symptoms, screening environmental samples for biothreat agents, as well as genotyping and whole-genome analysis of single pathogens.     

Crystal structure of human Gadd45 reveals an active dimer

The human Gadd45 protein family plays critical roles in DNA repair, negative growth control, genomic stability, cell cycle checkpoints and apoptosis. Here we report the crystal structure of human Gadd45, revealing a unique dimer formed via a bundle of four parallel helices, involving the most conserved residues among the Gadd45 isoforms. Mutational analysis of human Gadd45 identified a conserved, highly acidic patch in the central region of the dimer for interaction with the proliferating cell nuclear antigen (PCNA), p21 and cdc2, suggesting that the parallel dimer is the active form for the interaction. Cellular assays indicate that: (1) dimerization of Gadd45 is necessary for apoptosis as well as growth inhibition, and that cell growth inhibition is caused by both cell cycle arrest and apoptosis; (2) a conserved and highly acidic patch on the dimer surface, including the important residues Glu87 and Asp89, is a putative interface for binding proteins related to the cell cycle, DNA repair and apoptosis. These results reveal the mechanism of self-association by Gadd45 proteins and the importance of this self-association for their biological function.     

Functional proteomics in histone research and epigenetics

Post-translational modifications of histones comprise an important part of epigenetic gene regulation. Mass spectrometry and immunochemical techniques are currently the methods of choice for identification and quantitation of known and novel histone modifications. While peptide-centric mass spectrometry is a well-established tool for identification and quantification of histone modifications, recent technological advances have allowed discrete modification patterns to be assessed on intact histones. Chromatin immunoprecipitation assays (ChIP and ChIP-on-chip) are currently gaining tremendous popularity and are used to explore gene-specific patterns of histone modifications on a genomic scale. In this review, we introduce the basic concepts and recent developments of mass spectrometry, as well as immunochemical techniques and their applications in the analysis of histone modifications.     

Eight New Genomes and Synthetic Controls Increase the Accessibility of Rapid Melt-MAMA SNP Typing of Coxiella burnetii

The case rate of Q fever in Europe has increased dramatically in recent years, mainly because of an epidemic in the Netherlands in 2009. Consequently, there is a need for more extensive genetic characterization of the disease agent Coxiella burnetii in order to better understand the epidemiology and spread of this disease. Genome reference data are essential for this purpose, but only thirteen genome sequences are currently available. Current methods for typing C. burnetii are criticized for having problems in comparing results across laboratories, require the use of genomic control DNA, and/or rely on markers in highly variable regions. We developed in this work a method for single nucleotide polymorphism (SNP) typing of C. burnetii isolates and tissue samples based on new assays targeting ten phylogenetically stable synonymous canonical SNPs (canSNPs). These canSNPs represent previously known phylogenetic branches and were here identified from sequence comparisons of twenty-one C. burnetii genomes, eight of which were sequenced in this work. Importantly, synthetic control templates were developed, to make the method useful to laboratories lacking genomic control DNA. An analysis of twenty-one C. burnetii genomes confirmed that the species exhibits high sequence identity. Most of its SNPs (7,493/7,559 shared by >1 genome) follow a clonal inheritance pattern and are therefore stable phylogenetic typing markers. The assays were validated using twenty-six genetically diverse C. burnetii isolates and three tissue samples from small ruminants infected during the epidemic in the Netherlands. Each sample was assigned to a clade. Synthetic controls (vector and PCR amplified) gave identical results compared to the corresponding genomic controls and are viable alternatives to genomic DNA. The results from the described method indicate that it could be useful for cheap and rapid disease source tracking at non-specialized laboratories, which requires accurate genotyping, assay accessibility and inter-laboratory comparisons.     

Loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata infection in China targeting the 18S rRNA and ITS sequences

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria annulata, an economically important cattle disease in China that occurs in subtropical and tropical areas. These assays target the ribosomal RNA (18S rRNA) and ITS LAMP sequences. The primer set for each gene target consists of four primers, and each set recognizes six distinct regions on the target gene to allow for the highly specific detection of T. annulata. The specific ladder bands were amplified from the autologous genomic DNA of four Chinese-laboratory-preserved standard T. annulata stocks, and there were no cross-reactions with the genomic DNA of normal bovine blood and other protozoan species. The LAMP assays were sufficiently sensitive to detect 0.1 pg/μl of genomic DNA. Furthermore, DNA extracted from blood collected from cattle experimentally infected with T. annulata (18-105 days post-infection) was amplified, demonstrating the high sensitivity of these primers. Of the 351 field samples collected from China, 24.5% were positively detected by two LAMP primers, and 18.2% were found to be positive for T. annulata infection by PCR. These results indicate that the LAMP assay could be a potential diagnostic tool for epidemiological studies of T. annulata infection in China.     

Development and assessment of microarray-based DNA fingerprinting in <Emphasis Type="Italic">Eucalyptus grandis</Emphasis>

Development of improved Eucalyptus genotypes involves the routine identification of breeding stock and superior clones. Currently, microsatellites and random amplified polymorphic DNA markers are the most widely used DNA-based techniques for fingerprinting of these trees. While these techniques have provided rapid and powerful fingerprinting assays, they are constrained by their reliance on gel or capillary electrophoresis, and therefore, relatively low throughput of fragment analysis. In contrast, recently developed microarray technology holds the promise of parallel analysis of thousands of markers in plant genomes. The aim of this study was to develop a DNA fingerprinting chip for Eucalyptus grandis and to investigate its usefulness for fingerprinting of eucalypt trees. A prototype chip was prepared using a partial genomic library from total genomic DNA of 23 E. grandis trees, of which 22 were full siblings. A total of 384 cloned genomic fragments were individually amplified and arrayed onto glass slides. DNA fingerprints were obtained for 17 individuals by hybridizing labeled genome representations of the individual trees to the 384-element chip. Polymorphic DNA fragments were identified by evaluating the binary distribution of their background-corrected signal intensities across full-sib individuals. Among 384 DNA fragments on the chip, 104 (27%) were found to be polymorphic. Hybridization of these polymorphic fragments was highly repeatable (R²>0.91) within the E. grandis individuals, and they allowed us to identify all 17 full-sib individuals. Our results suggest that DNA microarrays can be used to effectively fingerprint large numbers of closely related Eucalyptus trees.     

Rapid typing of Coxiella burnetii

Coxiella burnetii has the potential to cause serious disease and is highly prevalent in the environment. Despite this, epidemiological data are sparse and isolate collections are typically small, rare, and difficult to share among laboratories as this pathogen is governed by select agent rules and fastidious to culture. With the advent of whole genome sequencing, some of this knowledge gap has been overcome by the development of genotyping schemes, however many of these methods are cumbersome and not readily transferable between institutions. As comparisons of the few existing collections can dramatically increase our knowledge of the evolution and phylogeography of the species, we aimed to facilitate such comparisons by extracting SNP signatures from past genotyping efforts and then incorporated these signatures into assays that quickly and easily define genotypes and phylogenetic groups. We found 91 polymorphisms (SNPs and indels) among multispacer sequence typing (MST) loci and designed 14 SNP-based assays that could be used to type samples based on previously established phylogenetic groups. These assays are rapid, inexpensive, real-time PCR assays whose results are unambiguous. Data from these assays allowed us to assign 43 previously untyped isolates to established genotypes and genomic groups. Furthermore, genotyping results based on assays from the signatures provided here are easily transferred between institutions, readily interpreted phylogenetically and simple to adapt to new genotyping technologies.     

Functional cell-based uHTS in chemical genomic drug discovery

The availability of genomic information significantly increases the number of potential targets available for drug discovery, although the function of many targets and their relationship to disease is unknown. In a chemical genomic research approach, ultra-high throughput screening (uHTS) of genomic targets takes place early in the drug discovery process, before target validation. Target-selective modulators then provide drug leads and pharmacological research tools to validate target function. Effective implementation of a chemical genomic strategy requires assays that can perform uHTS for large numbers of genomic targets. Cell-based functional assays are capable of the uHTS throughput required for chemical genomic research, and their functional nature provides distinct advantages over ligand-binding assays in the identification of target-selective modulators.     

炭疽芽胞杆菌疫苗研究进展

炭疽芽胞杆菌引起的炭疽病死亡率非常高 ,当前的疫苗具有效力不稳定、对吸入性炭疽的保护率低、免疫程序繁琐、存在副作用等缺点。近年来人们在改造传统疫苗的同时又有一些新的发现 ,如保护性抗原 (PA)的抗体在体内可杀死芽胞 ;通过粘膜免疫能够诱导机体分泌IgA抗体 ;抗多聚谷氨酸 (γ D PGA)抗体可以同炭疽杆菌的繁殖体作用 ,从而杀死繁殖体 ;寻找到新的免疫原。DNA疫苗、活载体疫苗的出现为新一代安全、免疫程序简单、具更高保护率的疫苗奠定了基础